^Program Director/Principal lnyestigatpr,(Last,,Firsl, Middle); HOchstraSSer, Mark PROJEGT'SUWMARY (See inslructioiis): ; T, . : EOkaryotes have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins, and often these proteins are then targeted for degradation by the proteasome. Substrates, include naturally short-lived regulatory factors and aberrant protein quality control (PQG) substrates. Many human disordeis, including neurodegenerativediseases such as AI?heinier'sand,Rarkinsbn's disease, diaBetes, cystic fibrosis, and certain cancers, are associated \withabhdr:malities in Lib-dependent proteolysis. The Ub-proteasome system presents promising drug targets for treating-these diseases. In this renewal, the PI proposes to extend studies On Ub-depehdent proteolysis, focusing onendpplasmic reticulum (ER)-associated degradation (ERAD) and basic features of membrane and nuclear protein ubiquitylation and.degradation. The proposed research will focus on the yeast Saccharomyces cerevisiae because of its experimental advantages and the fact that the Ub system in general, and the ERAD machinery in particular, is highly conserved Recent work has Identified a yeast Ub-ligase (E3) complex embedded in the ER and nuclear envelope membranes that is capable of recognizing a wide array of reguiatory and PQC substrates. This unusual complex includes a large integral membrane E3 called Doa10 and two Ub-conjugating enzymes (E2s), Ubc6 and Ubc7. Doa10 Is the prototype for a broadly conserved class of viral and eukaryotic Ub ligases. It was discovered from an analysis of a soluble nuclear substrate, the Mata2 transcription factor, but it also has membrane substrates. A second E3, Sxl5/Slx8, important for MATa2 degradation was also recently discovered. The overall goal of the proposal is to determine key mechanistic features of pi-otein ubiquitylation by the ER-membrane E3 ligases DoalO and Hrd1. We also hope to advance our currently very poor understanding of how membrane extraction of ER membrane substrates occurs in conjunction with theseE3s. For the soluble substrate MATa2, both its DoalO-dependent and Slx5/Slx8-dependent ubiquitylation will be explored. We expect to continue to gain important insights into fundamental aspects of Ub-proteasome system mechanism and function, including features unique to the PQC of membrane proteins at the ER. RELEVANCE (See inslrucllons): A smalt protein called ubiquitin regulates the growth of all human cells; it is attached to specific cel! proteins, and this.marks these proteins for destruction. Defects in enzymes controlling these processes cause neuro- degenerative disorders, developmental abnormalities, and cancer. This project aims to deepen our under- standing of enzymes that attach ubiquitin to damaged proteins and other proteins, with the long-term goal of llevalnninn therapies to treat natjents suffering from Alzheimer's diabete.':; cancer and other diseases PROJECT/PERFORMANCE SltE(S):: (if additlOT^^^ Project/Perfortnance Site Primary Location Organizational Name Yale University DUNS 0432075620000 Street 1 266 Whitney Avenue, BASS 224 Street2: P.O. Box 208114 City New Haven Couiity:;,, .,:,;,,.'; State: CT Province Country: USA Zip/Postal Code: 06520-8114